The present invention relates to methods for controlling yield of plants, in particular cereal crops, preferably maize crops, through the modulation of glutamine synthetase (GS) activity.
The cereals including maize, wheat and rice account for 70% of worldwide food production. When such crops are grown for protein content, they require large quantities of nitrogenous fertilizers to attain maximal yields. In the past few years, there has been considerable interest in nitrogen use efficiency (NUE), which can be defined as the kernel yield per unit of nitrogen (N) in the soil and the N utilization efficiency (NutE), which is the yield per N taken up (Hirel and Lemaire, A. Basra, and S. Goyal, eds. (Haworth's Food Product Press. Binghamton, New-York), 15: 213-257, 2005). A number of physiological and agronomic studies have been undertaken to identify which are the limiting steps in the control of N uptake, assimilation and recycling during plant growth and development (Jeuffroy et al., J. Exp. Bot., 53: 809-823, 2002) including cereals such as maize (Hirel et al., Physiol. Plant, 124: 178-188, 2005b), rice (Yamaya et al., J. Exp. Bot., 53: 917-925, 2002) and more recently wheat (Kichey et al., New Phytol., 169: 265-278, 2006). Using maize as a model crop, Hirel et al. (aforementioned, 2005b) have investigated the changes in metabolite concentration and enzyme activities involved in N metabolism within a single leaf, at different stages of leaf growth and at different periods of plant development during the kernel-filling period. It was concluded that total N, chlorophyll, soluble protein content and GS activity are strongly interrelated and are indicators that mainly reflect the metabolic activity of individual leaves with regards to N assimilation and recycling, whatever the level of N fertilization.
Glutamine synthetase (GS; E.C.6.3.1.2) catalyzes the conversion of inorganic nitrogen (ammonium) into glutamine, according to the following reaction:ATP+L-glutamate+NH3=>ADP+phosphate+L-glutamine.
All of the N in a plant, whether derived initially from nitrate, ammonium ions, N fixation, or generated by other reactions within the plant that release ammonium (decarboxylation of glycine during photorespiration, metabolism of N transport compounds and the action of phenylalanine ammonia lyase) is channeled through the reactions catalyzed by GS. Thus, this enzyme is likely to be a major check point controlling plant growth and productivity (Miflin and Habash, J. Exp. Bot., 53(370): 979-87, 2002).
In maize, the putative role of GS for kernel productivity has also been highlighted using quantitative genetic approaches. QTLs for the leaf enzyme activity have been shown to be coincident with QTLs for yield (Hirel et al., Plant Physiol., 125: 1258-1270, 2001), and a positive correlation has been observed between kernel yield and GS activity (Gallais and Hirel, J. Exp. Bot., 55: 295-306, 2004). Further QTLs for GS gene loci have been identified in relation to remobilization of N from the leaf, stem and whole plant, post-anthesis N uptake (Gallais and Hirel, aforementioned, 2004) and germination efficiency (Limami et al., Plant Physiol., 130: 1860-1870, 2002). The importance of GS in controlling cereal productivity has recently been strengthened by a study performed in rice, in which a strong reduction in both growth rate and kernel yield was observed in a mutant deficient in cytosolic GS (Tabuchi et al., Plant J., 42: 641-655, 2005).
GS in higher plants including maize can be readily separated by standard chromatographic, localization and western blotting techniques into cytoplasmic (GS1) and plastidic (GS2) forms (Hirel and Lea, In: Plant Nitrogen, P. J. Lea and J. F. Morot-Gaudry, eds (INRA, Springer), pp. 79-99, 2001). However, this distinction is not as simple as was first thought, as although only one gene has been shown to encode the plastidic form, a small family of up to five genes is now known to encode the cytoplasmic form (Cren and Hirel, Plant Cell Physiol., 40: 1187-1193, 1999). Initial experiments indicated that the five cytoplasmic GS genes were differentially expressed in the roots, stems and leaves of maize (Sakakibara et al., Plant Cell Physiol., 33: 1193-1198, 1992; J. Biol. Chem., 271: 29561-29568, 1996; Li et al., Plant Mol. Biol., 23: 401-440, 1993). GS1-2, which is an important GS isoenzyme of the developing kernel, is abundant in the pedicel and pericarp, but has also been shown to be present in immature tassels, dehiscing anthers, kernel glumes, ear husks, cobs and stalks of maize plants (Muhitch et al., Plant Sci., 163: 865-872, 2002; Muhitch, J. Plant Physiol., 160: 601-605, 2003). Compared to the four other genes encoding GS1, Gln1-5 is expressed at a very low level in leaves, roots and stems (Sakakibara et al., aforementioned, 1992; Li et al., aforementioned, 1993). In a more recent study of maize leaves carried out by Hirel et al. (aforementioned, 2005b), it was shown that two of the five genes encoding GS1 (Gln1-3 and Gln1-4) were highly expressed regardless of the leaf age and the level of N fertilization, although there was an increase in Gln1-4 transcripts in the older leaves. It has been suggested that Gln1-4 encodes a GS isoform that is involved in the reassimilation of ammonium released during the remobilization of leaf proteins, whereas Gln1-3 encodes a GS isoform, which plays a house-keeping role during plant growth (Hirel et al., aforementioned, 2005a). The plastidic GS (GS2) encoded by Gln2 was only expressed in the early stages of plant development, presumably to reassimilate ammonium released during photorespiration, which is at a much lower rate in a C4 plant compared with a C3 plant (Ueno et al., Ann. Bot., 96: 863-869, 2005).
Despite the information available, as outlined above, concerning the expression of the five cytosolic GS1 genes in maize and the evidence of their importance from the demonstration of QTLs, the precise effect of the individual GS1 isoenzymes in the plant phenotype and kernel production is not known.